In production of proteins, especially microbial production, the instability of these proteins during the production process, especially the fermentation process is of major concern to assure the profitability of the production.
When using recombinant techniques for the production of heterologous proteins it has been found that the protein product often forms aggregates which are recognizable within the cell as "inclusion bodies" (Williams et al., Science 215 (1982) 687-689).
With the accumulation of the protein in the cytoplasma or the periplasmatic space either as inclusion bodies or in an otherwise aggregated or complexed state, many proteins have been found to be protected against proteolysis or other modifications in the production organism.
In the fluid production medium protease inhibitors like bovine aprotinin have been added to protect heterologous products like insulin against proteolytic digestion in mammalian culture (.Johnson et al. in Kininogenases-kallikrein 4 eds. Haberland et al. (1977) 113-118, Schattauer-Verlag), and a similar result has been obtained by Novikov et al. (Biotech. Lett. 12 (1990) 547-550) with addition of synthetic serin proteinase inhibitors to the growth medium in a fermentation of a Bacillus subtilis strain expressing proinsulin.
Autoproteolysis has been shown to be a quite common event in biological systems. By use of an inhibitor of cysteine proteinases a higher level of the proteinases cathepsin B, H and C has been found in rats (Kominami et al., Biochem. Biophys. Res. Comm. 144 (1987) 749-756). Connor (Biochem. J. 263 (1989) 601-604) has shown that procathepsin D undergoes autoproteolytic change to produce the mature cathepsin D even at low concentrations (&lt;1 g/ml). In bovine heart the Ca-dependent protease II seems to be activated by autoproteolytic cleavage of a subunit and by further successive cleavages the Ca-dependence of the protease is lowered (Demartino et al., J. Biol. Chem. 261 (1986) 12047-12052).
In microorganisms autoproteolysis was shown to be the most likely cause of the maturation of a subtilisin type protease in Bacillus subtilis during the secretion of the proform (Power et al., PNAS 83 (1986) 3096-3100). Later studies by Zhu et al. (Nature 339 (1989) 483-484) and Egnell and Flock (Gene 97 (1991) 49-54) have shown that the proform functions to guide the subtilisin into the right conformation in order to achieve an autoproteolytic maturation.
With purified proteases autodigestion is a common cause of inactivation of the enzyme. Druckner and Botchers (Arch. Biochem. Biophys. 147 (1971) 242-248) have described the limited autodigestion of thermolysin from Bacillus stearothermophilus at high temperature and low calcium concentration. Van den Burg et al. (Biochem. J. 272 (1990) 93-97) have examined the autocatalytic degradation of a neutral protease from Bacillus subtilis and Kim et al. (Han'guk Saenghwa Hakhoechi 23 (1990) 58-61) have identified the major cleaving sites of subtilisin Carlsberg. Autoproteolysis might be a limiting factor in obtaining high fermentation yields of proteases.
During fermentations the problems concerning the proteolytic degradation of the products are traditionally minimized in adjusting the growth conditions such as temperature, pH and the amount of available nitrogen source or addition of protease inhibitors (International Patent Application No. PCT/DK 89/00194), but still the proteolytic degradation of the product very often will set the limitations for the yield.
On the other hand Coxon et al. (Letters in Appl. Microbiol. 12 (1991) 91-94) find that the use of a Bacillus subtilis strain deficient in extracellular proteases shows increased tendency to cell lysis as the cells approach stationary growth phase.
Therefore methods to minimize the contact between the product and the rest of the growth medium, especially proteases, during fermentation will be a valuable tool in process optimization. This will make it possible to use fermentation conditions which increase the productivity of the protein where it has no influence if more protease is produced. The use of a minimal medium gives better product quality as a major advantage, since it will result in an improved recovery process. However, the yield of protease is often rather low, which to a certain extent is ascribable to an increased tendency for autoproteolytic cleavage of the protease.
In this specification and the claims protein variants to be used or contemplated to be used in the present invention are described using the following nomenclatures for ease of reference:
Original amino acid(s) position(s) substituted amino acid(s)
According to this the substitution of Glutamic acid for glycine in position 195 is designated as: EQU Gly 195 Glu or G195E
a deletion of glycine in the same position is: EQU Gly 195 * or G195*
and insertion of an additional amino acid residue such as lysine is: EQU Gly 195 GlyLys or G195GK
Where a deletion is indicated an insertion in such a position is indicated as: EQU * 36 Asp or *36D
for insertion of an aspartic acid in position 36
Multiple variants are separated by pluses, i.e.: EQU Arg 170 Tyr+Gly 195 Glu or R170Y+G195E
representing a multiple variant "mutated" in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.